Hematopoietic stem cells (HSCs) are rare self-renewing cells in the hematopoietic compartment that fuel the production of millions of differentiated blood cells of various types. HSC transplantation has become a common clinical treatment for patients suffering from leukemia, aplastic anemia, sickle cell disease, and other disorders of hematopoietic systems. Because transplantation success depends heavily on the number of available HSCs in peripheral and cord blood resources, expansion of HSCs both in vivo and in vitro has assumed a high priority. Unfortunately, progress in this research area has been impeded by the limited understanding of the molecular mechanisms governing the decision of HSCs to self-renew or differentiate. In the proposed project, we aim to define the role of Slug, an evolutionarily conserved zinc-finger transcriptional repressor, in the regulation of HSC self-renewal. Our most recent work shows that Slug is dispensable for normal hematopoiesis, but is critical for suppression of blood cell repopulation after bone marrow transplantation or treatment with 5-fluorouracil (5- FU). Additional studies suggest that Slug is a potent negative regulator of c-kit and other cell cycle regulators (including cyclin D1, c-Myc, and Id3), while it is positively regulated by the SCF/c-Kit signaling in HSCs. These findings led logically to the central hypothesis of the current application: that Slug functions in a novel negative- feedback loop in SCF/c-kit signaling pathway, and that its deletion will promote symmetric division of HSCs during repopulation of the hematopoietic systems. These predictions will be tested by examining the effects of Slug deficiency on the integrity of HSCs following myelotoxic injury (Aim 1); by determining if Slug regulates symmetric cell division of HSCs during hematopoietic regeneration (Aim 2); and by elucidating Slug's role in the SCF/c-kit signaling pathway (Aim 3). Successful completion of these aims will be facilitated by innovative methods and strategies that were developed specifically for this project, including single cell division assays based on PKH2-26 dye and Notch-GFP reporters to discriminate between symmetric and asymmetric division, and a new HSC self-renewal competitive assay. The information we expect to generate will have the potential to improve HSC expansion both in vitro and in vivo.